Cancer is a genetic disorder in which a series of mutations subvert the normal developmental program of a cell and allow it to proliferate without constraint. Accumulation of deleterious mutations appear to represent the basis of cancer progression (Kinzler, K. W. and Vogelstein, B. Nature 1997 386:761-763). While these mutations can take many forms, the most characteristic form of genetic change in cancer cells is karyotypic instability with aneuploidy and chromosomal rearrangements, particularly balanced translocations. By joining together previously unlinked chromosomal arms, balanced translocations can result in the creation of hybrid genes with altered expression patterns for potential oncogenes or tumor suppressor genes. Karyotypic detection of translocations has been very useful for cancer researchers. Clinically, the presence or absence of specific translocations has therapeutic and prognostic implications. More fundamentally, genes identified at the translocation breakpoints are strong candidates for involvement in malignant transformation (Sanchez-Garcia, I. Annu. Rev. Genet. 1997 31:429-453). These translocations serve as markers of the malignant state and can be either the cause or the consequence of the transformed state. For example, the Philadelphia chromosome is a specific t(9;22)(q34;q11) translocation that fuses the B-cell antigen receptor gene BCR and the ABL oncogene (De Klein et al. Nature 1982 300: 765-767). This fusion is thought to represent the crucial event in the development of chronic granulocytic leukemia. However, this translocation can also appear later in the course of multiple forms of leukemia. In general, it appears that all hematologic malignancies originate from such "dangerous liaisons" between unlinked chromosomal segments. Solid tumors as well may have characteristic translocations, suggesting that the development of an unstable chromosomal state increases the likelihood of translocations which in turn increase the likelihood of tumor progression (Rabbitts, T. H. Nature 1994 372:143-149; Sanchez-Garcia, I. Annu. Rev. Genet. 1997 31:429-453).
While the identification and analysis of the genes present at specific translocation breakpoints has become an area of great research interest, causes of these translocations are still poorly understood. Clearly an understanding of possible causes, however, is important for cancer prevention, for identification of at risk individuals, and for developing potential targets of drug intervention.
Translocations are believed to arise by recombination, a descriptive term given to any process whereby double-stranded DNAs are broken and rejoined in ways that alter the linkage relationship of the genes near the breaks. At least three different recombination pathways that operate in cells which can cause translocations have been proposed.
First, during homology-dependent or homologous recombination, identical sequences on nonhomologous chromosomes are believed to crossover, resulting in new linkages of nonhomologous chromosome arms. This model is supported by the fact that repetitive sequences in the human genome such as Alu elements or retrotransposons such as LINE elements are occasionally observed at translocation breakpoints (Kato et al. Gene 1991 97:239-244). Studies from a number of model organisms, particularly Saccharomyces cerevisiae, indicate that the presence of a double strand break (DSB) greatly stimulates the process of homologous recombination, via strand invasion of a linear single-stranded end into complementary double-stranded sequences elsewhere in the genome (Petes et al. (1991) Recombination in yeast. "The Molecular and Cellular Biology of the yeast Saccharomyces". Broach, J. R., Pringle, J. R. and Jones, E. W., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Model systems for homology-directed transpositions have been developed in yeast and in mammalian cells which take advantage of the stimulatory effect of an induced DSB (Fasullo et al. Mutat Res. 1994 314:121-133; Haber, J. E. and Leung, W. Proc. Natl. Acad. Sci. USA 1996 93:13949-13954; and Richardson et al. Genes & Dev. 1998 12:3831-3842). However, it appears that homologous recombination is at best a minor pathway for translocation-formation in human cancers.
Site-specific recombination requiring a site-specific recombinase, as well as specific DNA recognition sequences, have also been proposed. In humans, B and T cell precursors go through a site-specific recombinational process known as V(D)J rejoining that is essential to their maturation. V(D)J rejoining assembles antigen receptor variable genes by making DSBs at specific recombination signal sequences and then rejoining non-contiguous intrachromosomal segments. Many reciprocal translocations associated with lymphoid malignancies involve a V(D)J cleavage-rejoining site at the breakpoint (Rabbitts, T. H. Nature 1994 372:143-149). This has led to the concept that these translocations result from aberrant nonhomologous rejoining events during this normally site-specific process. The V(D)J cleavage site represents one break. Potential sources of the second break include both physiological (e.g. transcription, replication, repair) and environmental (e.g. x-rays, free radicals). In these cases, the V(D)J break might be joined to another simultaneous break. It was recently demonstrated that the V(D)J recombinase can function as a transposase, capable of actively inserting cleaved DNA ends into random targets (Hiom et al. Cell 1998 94:463-470). Similar experimental systems using other site-specific recombinases have also been designed that can generate recombinase-dependent translocations in the presence of a recombinase and appropriate recognition sequences on separate nonhomologous chromosomes (Golic, K. G. and Lindquist, S. Cell 1989 59:499-509; Sauer, B. J. Mol. Biol. 1992 223:911-928; and Van Deursen et al. Proc. Natl. Acad. Sci. USA 1995 92:7376-7380).
Nonhomologous recombination is an inherently imprecise form of recombination that appears to be the major pathway for DSB repair in human cells (Meuth, M. (1989) Illegitimate recombination in mammalian cells. "Mobile DNA". Berg, D. E. and Howe, M. M., American Society for Microbiology, Washington, D.C.; and Roth, D. and Wilson, J. (1988) Illegitimate recombination in mammalian cells. "Genetic Recombination". Kucherlapati, R. and Smith, G. R., American Society for Microbiology, Washington, D.C.), although it represents a minor pathway in Saccharomyces cerevisiae (Haber, J. E. Bioessays 1995 17:609-620). In this form of recombination, no special sequences are present at the break sites. Instead variable length deletions or rearrangements have been observed at break sites. The recombination joints normally involve at most, a few (&lt;5) overlapping bases. Based on analysis of breakpoints, it is believed that most cancer-causing chromosomal translocations occur by nonhomologous end-joining of simultaneous DSBs that are present on separate nonhomologous chromosomes. This type of recombination has been studied in mammalian systems by analyzing sites of integration and excision of DNA viruses and transfected linear marker DNA, as well as by determining the genetic components required for the normal recombinational repair of V(D)J site-specific cleavage events (Roth et al. Current Biol. 1995 5:496-499). Further information has come from studies in Saccharomyces cerevisiae. The data so far suggests that there may be multiple end-joining pathways which utilize (amongst other proteins) Rad50, Mre11, Xrs2, Ku70, Ku80, the DNA-dependent protein kinase, DNA ligase 4, and XRCC4p (Hendrickson, E. A. Am. J. Hum. Genet. 1997 61:795-800).
The underlying causes of the translocations found in human tumors are believed to result from physiological, genetic, and/or environmental conditions which increase DSBs or decrease their repair thereby predisposing the cells to translocation formation. For example, lymphoid malignancies have associated translocations involving V(D)J recombination sites (Finger et al. Science 1986 234:982-985). The fact that V(D)J rejoining is a normal developmental process limited to lymphoid precursor cells underscores the point that the presence of a DSB at a specific chromosomal site makes that site a hotspot for translocations. As another example, several rare recessive human disorders have been identified, including Bloom's Syndrome, ataxia telangiectasia, and Nijmegen Breakage Syndrome, whose hallmarks are chromosomal instability, hypersensitivity to DNA damaging agents, and early onset of a variety of malignancies. Analysis of the genes that are defective in these syndromes suggests that their normal functions are to minimize natural DSBs or to halt progression of the cell cycle until DSBs are repaired (Brown et al. Proc. Natl. Acad. Sci. USA 1997 94:1840-1845; Carney et al. Cell 1998 93:477-486; Chaganti et al. Proc. Natl Acad. Sci. USA 1974 71:4508-12; Ellis et al. Cell 1995 83:655-666; Epstein et al. Medicine 1966 45:177-221; Kasten et al. Cell 1992 71:587-597; Krepinsky et al. Human Genetics 1979 50:151-6; Varon et al. Cell 1998 93:467-476; Watt et al. Genetics 1996 144:935-945; and Yamagata et al. Proc. Natl. Acad. Sci., USA 1998 95:8733-8738). In their absence, higher levels of DSBs can accumulate and be anomalously rejoined. Ionizing radiation is a well known environmental factor that damages DNA, in part by causing DSBs. The occurrence of leukemias and other malignancies in individuals exposed to high levels of radiation underscores this connection. More commonly, individuals treated with a wide range of cancer chemotherapeutic agents are at high risk for development of treatment-related malignancies, particularly acute myeloid and acute lymphoblastic leukemias (Rowley et al. N Engl J Med 1996 334: 601-603).
The presumed mechanism of action of many anticancer drugs is that they cause multiple DSBs, thereby triggering apoptosis. One particularly interesting class of drugs are the epipodophyllotoxins which target topo II. Topo II untangles long strands of DNA by cleaving double-stranded DNA via a covalently-bound intermediate, allowing passage of an intact DNA duplex through this break, and then rejoining the cleaved ends. Anti-topo II drugs that are associated with treatment-related leukemias are thought to function by stabilizing the broken DNA state and inhibiting the rejoining step. Thus, while accumulation of enzyme-induced DSBs may lead to apoptotic cell death in some cells, it may also result in reciprocal translocations in others.
Thus, these observations support a general model of translocation formation involving aberrant repair of DSBs. However, with respect to cancer causing translocations, it is still unknown whether DSB formation and subsequent rejoining is essentially a random process or whether factors influence the susceptibility of certain sequences and chromosomal regions to breakage and rejoining. The identification of factors which influence the likelihood of a translocation to occur at a specific chromosomal locus will lead to approaches to prevent specific rearrangements and to identify them at an early stage.
A hamster cell system has been designed in which the recognition sequence for the rare cutting endonuclease I-SceI was placed in the second intron of a hemizygous adenine phosphoribosyltransferase (APRT) gene (Sargent et al. Mol. Cell. Biol. 1997 17:267-277). In this hamster cell system, a constitutive I-SceI expression vector was used to generate cleavages at the cut site, and aprt- clones were recovered by growth on 8-aza-adenine. Using this system, illegitimate recombination events were detected. However, chromosomal rearrangements associated with the illegitimate recombination events were not characterized in these experiments.
In the present invention, an assay is provided for detecting nonhomologous translocations in eukaryotic cells including yeast and mammalian cells.